The present invention is based on the surprising discovery that the diphtheria toxin fragment A can be selectively targeted to certain mammalian cell types by introduction into the cell of chimeric toxin genes in which expression of a toxin fragment A coding sequence is controlled by mammalian cell-specific regulatory sequences. The toxin fragment A coding sequence is selectively expressed in the target mammalian cell, inhibiting protein synthesis and resulting in cell death. Cell-specific expression of such chimeric toxin genes was sufficiently restricted to effect selective killing of targeted cells without elimination of nontargeted cells. It was surprising that selective lethality could be obtained using such chimeric toxin genes because there was evidence that the introduction of a single molecule of fragment A into a cell would be lethal (Yamaizumi et al. (1978) Cell 15:245-250) and it was not known, prior to the present invention, if cell-specific regulation would be restricted enough to cause selective lethality.
Attempts have been made to use the diphtheria toxin A fragment to selectively kill undesirable cells, such as malignant cells, without destroying healthy cells. Such attempts have concentrated on replacement of the natural fragment B protein delivery mechanism with alternate delivery mechanisms based on the specificity of certain proteins for cell surface molecules, for example by preparing toxin fragment A protein conjugates with antibodies (immunotoxins), hormones or plant lectins.
In nature diphtheria toxin is synthesized and secreted by strains of Corynebacterium diphtheria which are lysogenic for bacteriophage .beta..sup.tox+. Diphtheria toxin inhibits protein synthesis in and is toxic to most eukaryotic cells that have been tested. The naturally occurring toxin, which is bacteriophage encoded, is a single polypeptide of about 58 kd (535 amino acids). The toxin is composed of two regions, separable by proteolytic cleavage, which are functionally distinct. Toxin activity is associated only with fragment A, the NH.sub.2 -terminal region of 193 amino acids. Fragment A functions by catalyzing the inactivation of eukaryotic elongation factor-2 (EF-2). The COOH-terminal 342 amino acid fragment B, is itself nontoxic, but functions to deliver the toxin fragment A to cells. Fragment A is nontoxic unless it is introduced into the cell cytoplasm. A review of the structure and function of diphtheria toxin is provided in Pappenheimer (1977) Ann. Rev. Biochem. 46:69-94.
The entire diphtheria toxin gene has been cloned and sequenced by separately cloning fragments having little or no toxic activity (Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA 80:6853-6857). The coding sequence of the mature toxin is preceded by a signal sequence which is believed to function in secretion of tox gene product (Kaczorek et al. (1983) Science 221:855-858). Several nontoxic mutant tox genes have also been cloned including tox45 (Leong et al. (1983) Science 220:515-517) which has a wild-type region A and nonfunctional B region, and tox228 (Kaczorek et al. (1983) supra) which carries mutations in both the A and B regions. Uchida et al. (1973) J. Biol. Chem. 248:3838-3844 and ibid. pp. 3845-3850 have identified several mutant DT proteins, including one with reduced toxicity (CRM176). The attenuated toxicity (about 90% of wild-type) of CRM176 results from a mutation which affects enzymatic activity of the tox176 fragment A.
Specific cellular DNA sequences which function in cell- or tissue-specific regulation have been isolated and identified in many cases. In most mammalian systems studied, cell-specific expression is mediated by an enhancer, a cis-acting DNA sequence, which is believed to selectively activate expression in a target cell in response to tissue or cell-specific trans-acting factors. Examples include immunoglobulin heavy chain (IgH) enhancers which are selectively active in B-cells (Gillies et al. (1983) Cell 33:717-728; Picard and Shaffner (1984) Nature 307:80-82; Ephrussi et al. (1985) Science 227134-140), control elements for elastase cell-specific expression (Hammer et al. (1987) Mol. Cell. Biol. 7:2956-2967), insulin (Edlund et al. (1985) 230:912-916) and interleukin-2 (Fujita et al. (1986)). It has been reported that cell-type specificity of immunoglobulin genes is conferred not only be the IgH enhancer but also by a 5'-upstream element associated with an immunoglobulin gene promoter, which upstream element appears to act independently of the IgH enhancer (Mason et al. (1985) Cell 41:479-487; Foster et al. (1985) Nature 315:423-425). A similar 5'-upstream promoter associated element is reported to function in insulin gene regulation (Edlund et al. (1985) supra) , but no such enhancer-independent promoter-associated element has been reported for interleukin-2 regulation (Fujita et al. (1986) supra).
Maxwell et al. (1986) Cancer Research 46:4660-4664 reports the selective killing of B-cells due to expression of an IgH enhancer-regulated diphtheria toxin fragment A gene and suggests that cell-specific regulatory mechanisms can be employed generally for selective cell killing by expression of a toxin gene, with applications in cancer therapy. Maxwell et al. (1987) Mol. Cell. Biol. 7:1576-1579 describes the cloning and sequencing of the attenuated diphtheria toxin 176 and suggests the use of the tox176 coding region for selective cell killing.
Palmiter et al. (1987) Cell 50:435-443 reports that a chimeric diphtheria toxin A coding sequence expressed under the regulatory control of elastase I enhancer/promoter sequences was selectively expressed in pancreatic acinar cells. Selective expression and selective lethality of the chimeric toxin gene was demonstrated by the production of transgenic mice lacking a normal pancreas. Breitman et al. (1987) Science 238:1553-1555 reports that expression of diphtheria toxin fragment A coding sequences under the control of gamma crystallin gene regulatory sequences resulted in selective elimination of lens tissue in transgenic mice.
Proposed treatments for Acquired Immune Deficiency Syndrome (AIDS) include "intracellular immunization" a term coined by Baltimore (1988) Nature 335:7395-7396) to describe the genetic modification of cells to render them incapable of supporting viral production. The present inventors have explored the use of regulated expression of the diphtheria toxin A fragment coding sequences and the attenuated toxin fragment gene tox176 to selectively kill cells infected with human immunodeficiency virus (HIV-1).
As described herein, the present inventors have placed expression of the luciferase (luc) reporter gene, or of diphtheria toxin fragment A coding sequences, under control of the HIV-1 trans-acting, essential Tat and Rev proteins. The Tat protein acts on a cis-acting element mapped to region +14 to +44 (referred to as the TAR region) of the HIV long terminal repeat (LTR) to increase viral expression from the LTR (Arya et al. (1985) Science 229:69-73; Rosen et al. (1985) supra; Sodroski et al. (1985) supra; Green et al. (1989) Cell 58:215-223). The Tat protein appears to exert effects at both transcriptional (Peterlin et al. (1986) Proc. Natl. Acad. Sci. USA 83:9734-9738; Hauber et al. (1987) Proc. Natl. Acad. Sci. USA 84:6364-6368; Laspia et al. (1989) Cell 59:283-292) and post-transcriptional levels (Cullen (1986) Cell 46:973-982; Feinberg et al. (1986) Cell 46:807-817; Wright et al. (1986) Science 234:988-992; Braddock et al. (1989) Cell 58:269-279; Edery et al. (1989) Cell 56:303-312). Tat can stimulate expression of heterologous genes placed 3' to the TAR region (Tong-Starksen et al. (1987) Proc. Natl. Acad. Sci. USA 80:6845-6849; Felber and Pavlaskis (1988) Science 239:184-187). The Rev protein relieves the negative regulatory effect of cis-acting repressive sequences (crs) found in the env region of the HIV-1 genome (Rosen et al. (1988) Proc. Natl. Acad. Sci. USA 85:2071-2075; Hadzopoulou-Cladaras et al. (1989) J. Virol. 63:1265-1274) which repress the production of viral unspliced and singly spliced messenger RNAs (mRNAs). The Rev protein acts by binding to RNA at the Rev responsive element (RRE; Malim et al. (1989) Nature 338:254-257; Cochrane et al. (1990) Proc. Natl. Acad. Sci. USA 87:1198-1202), also localized to the env region; binding of the Rev protein to the RRE is essential for Rev function. Rev protein expression results in an increased accumulation of unspliced and singly spliced viral mRNAs, encoding structural proteins, in the cytoplasm (Felber et al. (1989) Proc. Natl. Acad. Sci. USA 86:1495-1499; Zapp and Green (1989) Cell 58:215-223). Thus, expression of the Rev protein promotes the transition from early to latent infection to productive infection. Like Tat, the Rev protein can also act in trans to activate expression of heterologous genes which contain the negative crs sequences and a correctly oriented RRE (Rosen et al. (1988) supra; Felber et al. (1989) supra). The inventors demonstrate herein that efficient regulation of both chimeric luc and chimeric diphtheria toxin fragment A expression by the Tat and Rev proteins can be achieved in transfected cells in vitro. Such regulation is applicable in the treatment of AIDS, exploiting the extreme toxicity of diphtheria toxin fragment A or of the attenuated tox176 fragment A to kill virus-infected cells.